The glycosylceramidase in the murine intestine. Purification and substrate specificity.
The intestinal glycosylceramidase of the mouse which we reported previously as a taurodeoxycholate-activated galactosylceramidase (Kobayashi, T., and Suzuki, K. (1981) J. Biol. Chem. 256, 1133-1137) has been purified to homogeneity. The enzyme gave a single band of a molecular weight of 130,000 in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight estimated by Sepharose 4B or Sephadex G-200 gel filtration under nondenaturing conditions was 290,000 to 300,000. In the double immunodiffusion test, rabbit antiserum raised against the purified enzyme gave a single precipitin band against the enzyme, but no cross-reactivity was observed against the brain or kidney galactosylceramidase (EC 3.2.1.46). The purified enzyme was active toward 4-methylumbelliferyl beta-D-galactoside, beta-D-glucoside, beta-D-xyloside, beta-D-fucoside, and alpha-L-arabinoside. Among potential glycolipid substrates, the enzyme was active, in the presence of sodium taurodeoxycholate, toward galactosylceramide, glucosylceramide, lactosylceramide, galactosylsphingosine, and glucosylsphingosine. It was inactive toward GM1 ganglioside, asialo-GM1 ganglioside, desialylated fetuin, and desialylated transferrin. Among disaccharides, the enzyme showed the highest catalytic activity toward lactose (18.9 mumol/min/mg of protein) and the lowest toward galactose beta (1 leads to 4)-N-acetylglucosamine (0.06 mumol/min/mg of protein). Galactose beta (1 leads to 6)-N-acetylglucosamine was not hydrolyzed. Phlorizin was also a substrate for the enzyme.
D000818:Animals; D000936:Antigen-Antibody Complex; D005260:Female; D005959:Glucosidases / Q000302:isolation & purification*; D007118:Immunoassay; D005779:Immunodiffusion; D007421:Intestine, Small / Q000201:enzymology*; D007700:Kinetics; D010694:Lactase-Phlorizin Hydrolase / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008970:Molecular Weight; D013379:Substrate Specificity
D000936:Antigen-Antibody Complex; D005959:Glucosidases; D010694:Lactase-Phlorizin Hydrolase